The present invention is directed to a neuroprotectant composition wherein the active ingredient is pGLU-GLU-PRO-NH2 or a combination of pGLU-GLU-PRO-NH2 (EEP) and N-tert-Butyl-xcex1-(2-sulfophenyl)nitrone (SPBN). The present invention is also directed to a method of treating and preventing diseases and injuries of the brain, spinal cord and retina by administering the endogenous tripeptide EEP to a subject as a neuroprotectant or by administering EEP in combination with SPBN or other nitrone.
Twenty percent of all combat wounds involve the head. Penetrating head wound have a greater than 50% risk of developing posttraumatic epilepsy. Closed head injuries are more prevalent in the military than in the civilian community, and certain groups (e.g. paratroopers) are at special risk for both head and spinal injury. Among Naval personnel, certain occupational specialties are at increased risk for air gas embolism or decompression sickness (DCS), and the major neurological complication of either is spinal cord damage. There is also risk of oxygen toxicity-induced seizures. Severe head injury, or cerebral ischemia, is associated with a high mortality rate and poor functional outcome. Despite extensive clinical and experimental research, there are no well-defined therapies for these conditions. There are very few available treatments for brain injury today and the gradual progressive biochemical changes which occur after head trauma can lead to the evolution of permanent neuronal damage.
Further, personnel in all branches of the U.S. military are at risk for laser injury to the retina. Laser energy could be deliberately directed at the cockpit of U.S. military aircraft (airplanes and/or helicopters) with the intent of impairing the vision of pilots and/or crews (door gunners or medics). The adverse effects on the retina may range from transient impairment which can impact operational performance, to lasting disability or blindness. Reconnaissance troops and TOW missile operators are also at risk. Other potential sources of laser-induced retinal injury include exposure to laser targeting and ranging devices. A common source of laser-induced retinal injury in soldiers is the hand held neodymium (Nd:YAG) laser target designator (range finder), which operates at a wavelength of 1064 nanometers. Ruby lasers operating at a wavelength of 694.3 nm have also been used in military range finders and represent an additional potential source of retinal damage. The normal function of the lens, focussing light onto the retina, also serves to concentrate incident laser energy, when exposure occurs. While the magnifying effect of the human lens on intraocular laser energy is large as much as 10,000 foldxe2x80x94the amplification in the case of a soldier using binoculars could reach as much as 106. The amount of laser energy reaching the retina is also directly related to pupil diameter. Thus, soldiers are at greater risk under dark-adapted conditions. Finally, the location of the laser-induced lesion is clinically very important, with foveal location being the most severe. Laser injuries near the fovea present a risk of penumbral spread over time to include the fovea.
Naval personnel are also at risk for decompression sickness (DCS) in at least two operational scenarios: (1) SEALS on extended underwater operations; (2) Submariners, during emergency evacuation, of crew from a submarine disabled on the continental shelf. Navy SEAL operations often require prolonged (e.g. up to 10 hours) dives at shallow depth (e.g. 40-60 feet), breathing high concentration oxygen, sometimes followed by brief excursions at greater depth. These personnel are at risk for air embolism, which can cause spinal injury, similar to that seen in decompression sickness (DCS), and oxygen toxicity-induced seizures.
Submarines could become disabled (DISSUB) on the ocean floor, requiring emergency evacuation of the crew. In this scenario, arrival of a rescue ship could take days, during which time crews in a disabled submarine could be exposed to hyperbaric conditions. To accomplish a DISSUB rescue expeditiously, a submersible rescue vessel would transport the crew to the surface in groups, potentially without decompression. Once on the surface, crew members may be required to wait for access to treatment in a limited-capacity recompression chamber on the surface. Because of the delay in treatment, some personnel might be at significant risk for neurological complications of DCS. Spinal cord injury is a relatively common sequela of DCS; recompression treatment is not always successful and if delayed, prognosis for recovery is poor. The prolonged delay before submarine crews might be brought to the surface, provides a significant window of opportunity for administration of a safe, well-tolerated prophylactic treatment to mitigate risk and severity of possible neurological complications of DCS.
Leading causes of blindness include: age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma and cataracts. The prevalence of diabetes in North America is estimated to reach almost 17 million by the year 2000. In cases of insulin-dependent diabetes mellitus (IDDM) with onset before age 30, the average prevalence of proliferative retinopathy (DPR) is estimated at 23%. In IDDM of more than 30 years duration, however, the incidence of DPR rises to 70%. DPR is the leading cause of new cases of blindness in the U.S., accounting for 12% of new cases annually. The prevalence of AMD increases from over 2% in the age group of 60-64 years to over 25% in the 75-80 year group. It is estimated that the prevalence of glaucoma in the United States will be 2.9 million by the year 2000, and that over 130,000 will have been blinded by this disease.
A major pathological mechanism in both AMD and DPR is retinal neovascularization. The mechanisms of DPR include excessive retinal vascular permeability, edema, ischemia and the principal causes of loss of vision are hemorrhage into the vitreous and/or retinal detachment. The only effective treatment known for either AMD or DPR is coagulation via exposure to focussed laser irradiation (Vinding, 1995). Laser photocoagulation in a grid pattern is an effective treatment for the macular edema seen in diabetic retinopathy, as well as the neovascularization.
Unfortunately, exposure of the retina to laser energy, whether therapeutic or accidental results in formation of scotoma and visual impairment. Even after therapeutic exposures, there may be immediate and progressive visual impairment, due to destruction of normal retinal cellular elements with subsequent spread of injury to adjacent retinal tissue. In one study, progressive enlargement of laser scars was found in 11 of 203 patients with diabetic retinopathy treated via laser photocoagulation in a grid pattern.
Since the retina contains neurons and axons and is part of the central nervous system, recent advances in understanding of mechanisms of neurotoxicity apply, as do the related advances in research on neuroprotective agents. Ganglion cells are the output neurons of the retina and axons projecting from these cells form the optic nerve and project to the lateral geniculate nucleus of the brain. In one study, 12 hours after laser exposure, ganglion cells in monkey retina were liquefied. Ganglion cells are also destroyed in glaucoma, and continue to perish even after institution of standard glaucoma therapy.
Currently, post-injury treatment of spinal injury is most likely to include administration of the steroid methylprednisolone for 24 or 48 hours to reduce swelling and inflammation. In patients with accident-related acute spinal cord injury, clinical outcome at six months was improved in those receiving treatment with methyl prednisolone within eight hours of injury, compared to placebo-treated (Bracken et al., 1990 and 1997). Unfortunately, there is some evidence that glucocorticoids (GCs) can exacerbate the excitotoxic phase of neural injury. Postulated mechanisms of GC-mediated synergy with excitotoxic effects of glutamic acid (GLU) include: (1) GCs inhibition of reuptake inactivation of synaptic GLU, thereby increasing synaptic GLU levels; (2) GCs inhibition of calcium effiux from the postsynaptic neuron (McEwen and Sapolsky, 1995). In fact, methylprednisolone has recently been proven to exacerbate laser-induced lesions of the retina (Schuschereba et al., 1997). Other possible candidates, such as drugs which block the effects of glutamate at its NMDA receptor or the associated ion channel are associated with unacceptable behavioral toxicity (Tricklebank et al., 1989).
Some prior compounds have shown an ability to have some neuroprotective capabilities. For instance, thyrotropin-releasing hormone (TRH), is a tripeptide comprised of the amino acids pyroglutamate-histidine-proline-amide. TRH has been shown to be neuroprotective, analeptic, anticonvulsant and antidepressive. TRH was initially recognized as a peptide released by the brain to stimulate secretion of thyroid-stimulating hormone from the pituitary gland. Subsequently, TRH was identified in many brain regions and associated with numerous physiological functions. When injected, TRH was found to have many behavioral effects, including, analeptic, anticonvulsant and antidepressant (Sato et al., 1984; Sattin et al., 1987). Its in vivo neuroprotective effects in animal models of both brain and spinal cord injury have been reviewed by Faden et al. (1989). The early promise of TRH, however, has not been realized because it has properties which severely limit its clinical potential. Because injected TRH is rapidly hydrolyzed by the enzyme pyroglutamylpeptidase II which is found in blood (Cockle, et al., 1994; Klootwijk et al., 1997), its bioavailability and hence its clinical potential has been severely limited. Another limitation on therapeutic use of TRH might be potential for excessive activation of the thyroid gland.
TRH is not a suitable neuroprotectant because it is hydrolyzed by serum enzyme thyroliberinase which limits its bioavailability. Also TRH elevates plasma levels of thyroid hormone, which could potentially be problematic.
Other neuroprotectants are nitrone based free radical traps such as xcex1-Phenyl-N-tert-butylnitrone (PBN) which offer an ROS scavenging mechanism which differes from vitamin E and other endogenous compounds (*Althaus et al., 1998). The nitrones react covalently with ROS to form stable nitroxides and as such have been used to measure ROS. They have also been shown to be neuroprotective against glutamate-induced toxicity in cultured neurons (Zeevalk et al. 1998) as well as in several rodent models of cerebral ischemia, including transient global ischemia (Oliver et al., 1990), transient (ZHO et al., 1994, Folbergrova et al., 1995) and permanent [100 mg/kg 30 mim pre-ischemia; repeated at intervals after injury] (Cao and Phillis, 1994) occlusion of the middle cerebral artery (Read et al., 1999). Unfortunately, PBN caused significant toxicity at high doses (Haseloff et al., 1997) and is thus, unsuitable for serious consideration as a neuroprotectant.
SPBN (N-tert-Butyl-xcex1-(2-sulfophenyl)nitrone) is also a neuroprotectant without significant toxicity and studies have shown that SPBN is protective against striatal injections of NMDA, kainic acid, AMPA, MPP+, 3-acetyl pyridine and malonate, decreasing the volume of the lesions induced by those toxins (Schulz et al., 1995), as well as decreasing malonate induced formation of ROS (Schulz et al., 1995).
Finally, another nitrone NXY-059, was shown to be effective in both tempory (Kuroda et al., 1999) and permanent focal ischemia models in the rat, and was effective when given 3 hours (Kuroda et al,1999) or even 5 hours (Maples et al., 1999) after start of recirculation in the former. In preliminary studies, NXY-059, administered either 15 min prior or 30 min post TBI was effective in decreasing volume of necrosis in the controlled cortical impact model (Cheng, et al., 1999). This compound is currently being developed for treatment of stroke by Centaur Pharmaceuticals and Astra/Zeneca.
Since no proven effective therapy for treating diseases and injuries to the brain, spinal cord and retina, are yet known, the importance of finding such therapeutic neuroprotectant is self-evident. What is needed is an effective neuroprotectant which has clinical use across a wide spectrum of injuries and diseases. What is also needed is a neuroprotectant which is not rapidly hydrolyzed in the blood and which is not toxic at low or high doses. Therefore, the object of the invention is to provide such a neuroprotectant to fill the current needs.
Briefly, this and other objects of the present invention as hereinafter will become more readily apparent.
The invention solves the above problems associated with known neuroprotectants by providing a neuroprotectant composition wherein the active ingredient is pGLU-GLU-PRO-NH2 (EEP) or a combination of pGLU-GLU-PRO-NH2 (EEP) and N-tert-Butyl-xcex1-(2-sulfophenyl)nitrone (SPBN). The present invention is also directed to a method of treating and preventing diseases and injuries of the brain, spinal cord and retina by administering the endogenous tripeptide EEP to a subject as a neuroprotectant or by administering EEP in combination with SPBN or other nitrone.